Chlamydia trachomatis is a widely disseminated, obligate intracellular pathogen that causes a range of diseases including trachoma, conjunctivitis and pelvic inflammatory disease. Within infected cells, C. trachomatis efficiently re-routes endocytic and exocytic traffic to create a growth-permissive compartment termed the Inclusion. Because Chlamydiae are not currently amenable to genetic manipulation, little progress has been made in identifying bacterial or host factors required to reprogram vesicular traffic in the host cell. Nonetheless, the increasing availability of Chlamydia genomic sequences provides the opportunity for the design of new experimental approaches to study Chlamydia pathogenesis. We propose a functional genomic approach to identify factors required for the biogenesis and maintenance of the C. trachomatis Inclusion. We propose to perform a three-stage screen of Chlamydia expression libraries in model eukaryotic systems to identify bacterial factors that interfere with endocytic traffic. Because C. trachomatis-mediated disruption of endocytic traffic is limited to the Inclusion, we hypothesize that the function of many Inclusion proteins is restricted to the surface of endosomal membranes. Therefore, a central aspect of our expression system includes anchoring Chlamydia proteins to the surface of endosomes (Endosomal Display). In the first screening stage, we utilize whole cell-based assays in the yeast Saccharomyces cerevisiae to rapidly sample all Chlamydia proteins of unknown function for factors that disrupt eukaryotic cellular functions. In the second stage, Chlamydia proteins that display a phenotype in yeast are expressed in Chinese Hamster Ovary (CHO) cells to confirm and/or extend phenotypes observed in yeast. We will raise antibodies to these Chlamydia proteins to determine their subcellular localization during Chlamydia infections. In the last stage of the screen, we will use the antibodies and cell lines generated to determine if these factors are necessary for Chlamydia growth in host cells. A third of the Chlamydia genome encodes proteins whose function cannot be determined from sequence analysis. We expect that the functional approach we propose will lead to the identification of novel classes of virulence factors and represent a major breakthrough in our understanding of Chlamydia pathogenesis.